Process for the separation of the hormone and hormonelike components of the pituitary gland



Patented Nov. 21, 1944 Q PROCESS 'FoR THE SEPARATION or THE HORMONE ANDHORMONELIKE Como: NENTS or THE PITUITARY GLAND Gerhard A. Fleischer andErwin Schwenk. Montclair, N. .L, assignors to Schering Corporation,Bloomfield, N. J a corporation of N ew Jersey No Drawing. ApplicationNovember 6, 1940, Serial No. 364,542

11 Claims.

The present invention relates to the extraction and separation of thediilerent groups of hormones and hormone-like substances of thepituitary gland.

This application is a continuation in part of our application Serial No.299,978, filed October 18, 1939, now Patent No. 2,327,375, issued August24', 1943.

It is the general object of the invention to provide an efiicient andeconomical process which is applicable to the whole pituitary gland,that is, to both the anterior and posterior lobes, for separating thehormone substances from the inert material and from each other; and moreparticularly to provide a process whereby the thyreotropic andgonadotropic hormones are isolated from the glandular material and fromeach other in such a manner that the residual material can beeffectively worked up for the recovery of other hormones andphysiologically active materials, including the lactogenic (prolactin)and corticotropic hormones and the hypnotic, oxytocic and vasopressorfactors.

It is a further object of the inventionto provide an improved processfor the isolation and separation of the hormones and hormone-likesubstances and other physiologically active components of either theanterior lobes or the pos terior lobes of the pituitary, or fromextracts prepared from either of the lobes. Other objects of theinvention will appear from the following detailed description.

It is known that the two lobes of the pituitary gland, the anterior lobeand the posterior lobe, contain a number of substances of hormone orotherwise physiologically active character which heretofore have beenisolated by processes resulting in the preparation of each of themseparately. The methods used will often destroy or impair someof theother active substances present in the pituitary and so make itimpossible to recover most of the active substances from the same batchof material. This is a fact of great importance, because thepituitaries, owing to the small size of the gland, are extremelyexpensive in comparison with other glandular starting materials. Amethod which will allow the preparation of several of the activesubstances from the batch of pituitaries must therefore be considered ofgreat commercial importance.

We have found that by using a modification oi the procedure described inour copending application Serial No. 299,978, the isolation of thedifferent active principles of the pituitary. from the same batch ofstarting material can be emciently accomplished. We have found that byadjusting the pH, salt concentrations and the concentrations andrelative amounts of the precipitating agents and reactingv materials, itis possible to separate the different active principles. As startingmaterial, whole pituitary glands may be used,

' but the process may also be used for the working up of the separatedanterior and posterior lobes of the pituitary glands. Furthermore, drypreparations may be used which are obtained when the finely groundpituitary glands, or the separated lobes, are dried in vacuum, or whensuch ground material is treated with high concentrations of acetone anddried afterwards.

Other starting materials include alkaline ex-.-

tracts precipitated with acetone or alcohol, as described, for instance,by Riddle and Bates (Bates, Robert W., and Riddle, Oscar: J. Pharmacol.and Exp. Therap. 55, 365 (1935)) and containing all of the hormoneprinciples. All these starting materials will behave similarly whensubjected to our new process.

We have found further that it is possible to divide the activesubstances present in the pituitary into three groups. The first groupconsists of such active substances as by virtue of their high molecularweight and composition are'very closely related to the proteins, as, forinstance, the lactogenic' hormone of the pituitary. Theadreno-corticotropic hormone of the anterior lobe behaves similarly. Asecond group comprises substances which, like the gonadotropic hormones,the synergist of Evans and coworkers and the thyreotropic hormone, haveapparently smaller molecular weights and in their composition probablyresemble or even are identical with the socalled glyco-proteins(mucoids). A third group comprises substances of. the type of theoxytocic and vasopressoric principles of the posterior lobe. These areprobably amines of not Very high molecular weights.; In this part of theextracts also a peculiar new substance is contained which acts as ahypnotic.

Our researches have established that the first group of physiologicallyactive substances can be separated from the other groups by adjustingthe pH value, preferably to between 5 and 6 (although higher values canbe used), and treating, for

' example, the finely ground whole glands, or the extracts from thewhole gland mentioned above, in aqueous suspension or solution with aninert Water-immiscible organic liquid like chloroform and other liquidsmentioned in the above-named copending application and preferably in thepresence of a salt. By shaking or stirring this mixsimple experiment.

ture where a sumcient volume of the water-immiscible liquid has beenemployed, three layers.

from the gel by centrifugaticn or otherwise, contains all the otheractive principles, including the thyreotropic and gonadctropic factors.By treatmerit of the water solution with organic solvents miscible withwater, such a methyl, ethyl, propyl, tertiary butyl, and otherwater-miscible alcohols, acetone, ethylene giycol and the like, theconcentration of this solvent being at least about 60%,

a precipitate is obtained which contains the gonadotropic hormones, thatis, the follicle stimulating and the luteinizing factors and thesynergist as well as the thyreotropic hormone. The concentration of theorganic sblvent depends to a certain degree upon the pH of the solution;with increasing pH value an increase in the amount of alcohol or similarsolvent becomes necessary. The optimum concentrations for any givenconditions can be readily determined by From the precipitate obtained inthis way, the crude hormones may be separated, for example, in themanner described below. The now remaining solution contains the thirdgroup of active substances mentioned above, namely, the oxytocic,vasopressoric, and hypnotic rinciples of the posterior pituitary lobe.

A further purification of the prolactin contained in the first-mentionedgel may be obtained by known methods. The only important impurities inthis gel are proteins of much higher molecular weight than the prolactinitself. For instance, by extracting the gel at a pH value of 8-10 withan aqueous solution of about (50-70% alcohol or about 60% acetone, theprolactin is quantitatively extracted, and after elimination ofthe inertproteins may be precipitated with a higher concentration of the-solventand a lower pH value. It is still contaminated with adrenocorticotropichormone which may be separated from the prolactin by iso-electricprecipitation.

tatlon of the gonadotropic hormone contain the substances of theabove-mentioned third group. 'Iheyarefreed from the alcohol oracetoneand then concentrated at very low temperature to about of theirvolume. This solution is then dialyzed. The oxytocicand vasopressoricprinciples dialyze together with the saltsl After dialyzation the innersolution may be used as such or may also be evaporated to dryness ingood vacuum at a temperature not exceeding 20. This material containsthe hypnotic factor. When i jected into animals as, for instance, miceor rats, the animals become drowsy and the temperature, as measured inthe rectum, drops considerably. After a certain time the animals recoverand the temperature reaches the normal level again.

By the term inert, water-immiscible organic liquid, as used herein, ismeant a substantially neutral organic water-immiscible liquid which isnon-reactive under the conditions of use and is capable of forming aphase surface of contact with water and of forming a gel with proteins,if necessary atter vigorous agitation. The solubility of such liquidshould preferably be not less than about 0.5% and not morethan about 10%in water, although somewhat higher solubilities may be found to besatisfactory in certain instances so long as the organic liquid forms adistinctly separate layer with water, the proportion of organic liquidbeing chosen accordingly. As suitable liquids of this type in additionto chloroform are methylene chloride, cyclohexanone, amyl alcohol,dichloro-ethylene, ethylene chloride, and mixtures of these, forexample, chloroform and amyl alcohol. The gel formation is preferablyaccomplished in an aqueous medium containing a The second group ofhormones is separated and purified by treatment of the solutioncontaining gonadotropic hormone and thyreotropic hormone with picricacid, or similar substances, like pic'- rolonic acid, or otherprecipitating agents for the thyreotropic hormone, By treating thesolution with picric acid at a saturation of not more than 75%, aprecipitate is obtained which contains practically all of thethyreotropic hormone and is substantially free from the goiadotropicfactor.

'From the supernatant liquid the gonadotropic hormones are precipitatedafter neutralization by the addition of alcohol or acetone to at least iseparation of the FSH and the ICSH factors (folhole-stimulating hormoneand interstitial-cellstimulating hormone).

small amount of a salt, which salt may be either added as such, orformed on the acidification of an aqueous alkaline extract of theglandular material. Where it is present, the amount of salt should besuflicient to create the particular electrical charge necessary to theformation of a gel, but it should'not, be of such'nature or present insuch quantities is to prevent the precipitation of the hormone and othersubstances which, as described below, are carried down in the gelprecipitate; nor should the quantity of salt employed be such as tointerfere with the subsequent separation of the three layers that areformed (aqueous layer, water-immiscible solvent, and gel).

The initial extraction of the glandular material is preferably carriedout with a solution of an inorganic base, for example, of sodium orpotassium hydroxide or carbonate, ammonium hydroxide, lithium hydroxide,barium hydroxide and the like. Organic bases may, however, also beemployed, such as pyridine, trimethylamine, urea, and other bases. Theinitial extraction can also bev efl'ectedunder slightly acid conditions,as with water acidified with acetic, lactic, phosphoric, and other,preferably relatively weak,

acids. The agitation with the water-immiscible The mother liquorsobtained from the precipiso should, of course, be chosen with therelative solubilities in mind to the end that two definite and separablelayers as between the water and organic liquid are maintained. Whennecessary, the volume of the aqueous extract may be reduced, as bypartial evaporation at low temperatures in vacuo. In any event, theamount of salt material present, as already indicated, should not be sohigh as to interfere with the separation of the water and the organicliquid into distinct layers.

The salts employed during the gel formation and subsequently to effectseparation of various components include the acetates, sulphates, andchlorides of sodium, potassium, lithium, magnesium, and ammonium,calcium chloride, and other water-soluble salts which in general do notform chemical combinations with the hormones or'other physiologicalcomponents of the mixture.

The invention is described in greater detail in the following exampleswhich are presented by way of illustration only.

Example 1 4.46 kg. of frozen undissected sheep pituitaries are groundtwiceand then stirred for hours with 13.38 liters of water +4213 cc. ofNaOH. The pH of the extraction is then adjusted to 5.5 with acetic acid.8.92 liters of chloroform are added and this mixture is shaken orstirred vigorously for one hour and then centrifuged. The clear aqueoustop layer is decanted and called A-l. The remainder consists of a geland a liquid layer, the latter consisting of chloroform which afterdistillation, whereby a fatty residue is obtained, may be used again inthe process. The gel, which we shall call A, is then stirred intoacetone and collected on a filter. When dry, it is extracted for 4 hourswith a mixture of 8.5 liters of alcohol and 4.28 liters of water at pH9.5, the pH being adjusted with 20% NaOI-I. This is centrifuged and theresidue re-extracted for the second time with a mixture of 8.5 liters ofalcohol and 4.28 liters of water, this time at pH 10.0. The insolublematerial which consists of inert proteins is then discarded.

The two extractions are combined, adjusted to approximately pH 6 bymeans of acetic acid and precipitated with 2 vols. of acetone. Thisprecipitat is allowed to settle in the ice box over night. The next dayit is worked up by syphoning off the supernatant liquor and filteringthe residue. The product thus obtained consists of prolactin besides thecortiootropic hormone and some other material. It is stirred for onehour acetone. It is a white powder, assaying..12 Riddle units ofprolactin per milligram. It does not have any toxic or anaphylacticproperties.

The solution A-1 is precipitated with 5 vols. of alcohol or acetone. Theprecipitate, called B, is worked up the next day and yields around 80grams of a preparation containing all gonadotropic factors, thethyreotropic hormone, and the synergist. The supernatant liquid 13-1 isevaporated in vacuum at an inside temperature of 20 C. or less, to about1.5 liters. It is then dialyzed to remove all salts and the oxytocic andvasopressoric principles and is concentratedfurther in vacuo to about200 cc. This solution constitutes a very potent extract of the hypnoticfactor.

Precipitate B is dissolved in 20 parts of water. It is then acidified topH 3.0 with dilute hydrochloric acid and picric acid is added to aconcentration of 0.6%, which is 50% of saturation. The precipitate isallowed to settle in the ice-box over night. The next day thesupernatant liquid is separated by syphoning and centrifuging. It isdesignated C-1 and contains thegonadotropic principles and thesynergist, whereas the precipitate 0 contains the thyreotropic hormone.The latter is separated from the picric acid by taking it up in dilutedammonia so that the pH is about 7.5. It. is precipitated with 5 parts ofacetone, the precipitate collected on a filter and washed with acetoneuntil free from picric acid. The yield is about 30 grams of thyreotropichormone which ca be further purified by'presolution with zincgonadotropic hormone and synergist, called D,

whereas the mother liquor D-l contains only some inert material. Theseparation of the thyreotropic from the gonadotropic hormones in theaqueous solution'of precipitate B may in general be conducted along thelines described in the application of Hans Maier-Huser and KarlJunkmann, Serial No. 250,160, now Patent No.

with 30 parts by weight of acetone and 1.5 parts .properties, thesupernatant liquid containing all the lactogenic and adrenocorticotropicactivity. The corticotropic hormone is obtained as a precipitate byadjusting the'pH of the solution to 6.5 and the salt concentration to4%. The supernatant liquor is thenadjusted to pH 5.5 and 15% sodiumchlorine content. The precipitate so formed is separated from thesolution, washed first with 15% sodium chloride solution, then with alittle ice-cold water, and finally dried with 2,331,334, issued October12, 1943.

The separation of thyreotropic and gonadotropic hormones can also becarried out directly with solution A-l, that is, without preliminaryseparation of the hypnotic, oxytocic, and vasopressor factors. In thiscase the mother liquors of the gonadotropic hormone contain not only thehypnotic, oxytocic, and vasopressor principles but also picric acid. Thelatter, however, crystallizes out and such solutions are concentrated invacuo and the last traces 0f picric acid can be removed by shaking outthe concentrate with amyl alcohol.

Example 2 added and the mixture is shaken vigorously for 1 with, forexample, picric acid in order to prehour with 750 cc. of methylenechloride. It is then centrifuged. The water layer is saturated cipitateboth the gonadotropic and thyreotropic hormones. 'They may be separatedaccording to Example 1. The methylene chloride gel is freed of a muchmethylene chloride as possible and is then dried by treating it withacetone. The

temperature should, preferably, at no time exceed 20 C. It is furtherpurified according to Example 1. p

Example 3 500 grams of frozen posterior pituitaryglands are maceratedand are extracted with 1.5 liters of water at pH 7.5. After stirring hasbeen continued for 4 hours, the mixture is filtered and the pH value ofthe filtrate is adjusted to 4.0 by the addition of acetic acid, and 1.5liters of chloroform plus 50 cc. of amyl alcohol are then added. Thismixture is stirred or shaken vigorously for 1 hour and then centrifuged.The clear supernatant water layer is decanted, whereas the chloroformgel is dried as described above. The volume of the water extract is thenreduced to about d cc. and this is put in a dialyzer and is dialyzed for43 hours. After dialyzing has been completed, a separation of theposterior hormones has taken place, the inner solution containing thehypnotic factor whereas the outside solution contains the oxytocic andvasopressorlc hormones. The latter can be worked up according to knownmethods. The solution containing the hypnotic factor is againconcentrated in vacuo, and centrifuged, and may be used as such orfurther purified.

As will be evident to those skilled in this art, variations from thespecific procedures, concentrations, and pH values may be resorted to,if necessary, with compensating change in other conditions, withoutdeparting from the principles of the invention. Thus the treatment withthe water-immiscible liquid may be conducted at a pH value lower than5.5, or it may take place in the alkaline range, say at a pH value ofabout 8.0. The salt employed during this treatment may be of varioustypes and its concentration. will be adjusted in known manner' for anyparticular extract to produce the desired separation with the highestefliciency.

We claim:

1. Process for the separation of the lactogenic and corticotropichormones from pituitarygland material, which comprises treating suchmaterial -in an aqueous acid medium with a waterimmiscible liquidcapable of forming a gel with the protein-like substances of highmolecular weight, extracting the gel with a mixture of water and awater-miscible organic liquid under alkaline conditions, increasing theconcentration of organic solvent and reducing the pH to a value below '7to cause precipitation of the lacsolution to a higher value at a pHvalue of approximately to eifect the precipitation of the corticotropichormone, and reducing the pH value .of the separated residual solutionand increasing the salt concentration to eiTect precipitation of thelactogenic hormone.

5. Process for the separation of the lactogenic and corticotropic'hormones which comprises treating an aqueous solution of the twohormones at low acidity with a salt'solution sufficiently concentratedto effect precipitation only of the corticotropic hormone, andthereafter increasing the acidity and likewise the concentration of saltto cause precipitation of the lactogenic hormone.

6. Process for the isolation and separation of the lactogenic andcorticotropic hormones from pituitary extracts, which comprises treatinga suspension of pituitary gland material under acid conditions withchloroform and agitating the mixture until a gel is produced, separatingthe gel, extracting the same with a mixture of water and alcohol at analkaline pH, treating the extract at a pH value of about 6 with acetoneto increase the concentration of organic liquid sufllciently to causeprecipitation of the lactogenic and corticotropic hormones, purifyingthe precipitate by extraction with acetone and dilute mineral acid,extracting the solid residue with an approximately 1% NaCl solution,raising the pH value of the solution to about 7.5, separating theprecipitate, reducing the pH value of the solution to about 6.5 andincreasing the salt concentration to about 4%, separating theprecipitate of the corticotropic hormone, reducing the pH value of theliquor to about 5.5 and increasing the salt concentration to about 15%,and separating the resulting precipitate of the lactogenic hormone.

7. In a process for the recovery of physiologically active components ofthe anterior lobe of the pituitary gland, the steps which compriseadding a salt to an aqueous extract of such lobe, agitating the mixtureunder acid conditions with an inert water-immiscible organic liquidcapable of forming a gel with protein and protein-like substances untila gel is formed, and separating the aqueous solution from the gel.

8. In a process for the recovery of physiologically active components ofthe posterior lobe Y of the pituitary gland, the steps which comprisetogenic and corticotropic hormones, and subsequently separating suchhormones.

2. Process according to claim 1, wherein the lactogenic andcorticotropic precipitate is treated with a dilute salt solution toeffect solution of the lactogenic and corticotropic hormones, and 1 thepH value of the extract then raised to about 7.5 to effect precipitationof impurities.

3. Process according to claim 1, wherein the lactogenic andcorticotropic precipitate is extracted with an aqueous medium to bringthe lactogenic and corticotropic hormones into solution and then addingsalt to the solution and adjusting the pH value to approximately 6.5 toeffect the precipitation of the corticotropic hormone.

4. Process according to claim 1, wherein the lactogenic andcorticotropic precipitate is treated with a dilute 'salt solution tobring the lactogenic and corticotropic hormones into solution, thenadjusting the salt concentration of the agitating an aqueous extract ofsuch lobe at a pH value of about 5.5 with an inert waterimmiscibleorganic liquid capable of forming a gel with protein and protein-likesubstances until a gel is formed, separating the aqueous solution fromthe gel, extracting the gel with a mixture of water and a water-miscibleorganic solvent under alkaline conditions, and successivelyprecipitating the corticotropic and the lactogenic hormone byprogressively increasing the hydrogen ion concentration and theconcentration of an added salt.

9. In a process for the separation of the physiologically activecomponents of the pituitary gland, the steps which comprise agitating anaqueous acidified suspension of pituitary gland material with an inertwater-immiscible organic liquid capable of forming a gel with .part ofthe components of said material until a gel is formed, separating theaqueous layer from the gel, extracting the gel, under alkalineconditions, with an aqueous medium, and precipitating the lactogenic andcorticotropic hormones from the said aqueous medium by treating thelatter, under increased hydrogen ion concentration, with awater-miscible organic liquid.

10. In a process for the separation of the lactogenic hormone frompituitary gland material 4 liquid'capable of forming a gel with proteinsand protein-like substances untila gel is formed, re-

moving the gel containing the lactogenic hormone, and separating thehormone from the gel. 11. Process according to claim 10, wherein thepituitary gland material is agitated with'chloroform at a pH value ofabout 5 to 6.

GERHARD A. FLEISCHER. ERWIN SCHWENK.

